Ng tosyl arginine and peptide synthesis therewith



United States Patent 3,131,174 N TGSYL ARGININE AND PEPTIDE SYNTl-ESHSTHEREWHH Robert Schwyzer, Riehen, Switzerland, assignor to CibaCorporation, a corporation of Delaware No Drawing. Filed Aug. 19., 1959,Ser. No. 834,635 Claims priority, application Switzerland Sept. 5, 19585 Claims. (Cl. 260-112) The present invention relates to the temporaryprotection of the amino groups in arginyl compounds, that is to say ofthe a-amino group and of the guanidino group of the arginyl radical ofthe formula NH 1TIH--C ((3 2):: Hui-015F00- in compounds containing saidradical, inter alia in arglnine itself or in arginyl-aminocarboxylicacids and derivatives thereof, more especially in peptides.

It is known that the arginyl radical is present in various peptidesoccurring in nature. The synthesis of peptides containing thisaminocarboxylic acid radical is, therefore, of considerable importance.In such syntheses the guanidino group must be protected since itotherwise would undergo undesirable acylations. Hitherto, the nitrogroup has been used as protecting group; this group can be eliminated byhydrogenation, but this is accompanied by the elimination of a radicalthat can be split off by hydrogenation, such as the carbobenzoxy group,for protection of the a-amino group. It is thus in this case impossibleto achieve the liberation of a protected amino group in the tit-positionby the handy and mild method of catalytic hydrogenation as this would beaccompanied by elimination of the nitro group.

Moreover, the above-mentioned method of protecting the guanidino groupby means of the nitro group is unsuitable in cases where the guanidinogroup cannot be liberated by hydrogenation, for example when the peptidecontains a sulfur compound.

As an alternative method of protecting the guanidino group it has beenproposed to make use of the carbobenzoxy group which can be eliminatedboth by hydrogenation and by hydrolysis. This acyl radical is, however,not adapted to prevent further acylation taking place at the guanidinogroup during the synthesis of the peptide.

The present invention is based on the observation that the amino groupsof the arginyl radical can be temporarily protected with advantage byusing as protection for the guanidino group an arylsulfonyl group andfor the ocamino group a radical capable of elimination by hydrogenation.

The aryl radicals may belong to the monocyclic or dicyclic series, suchas the benzene or napththalene series. Advantageously, they are radicalsof the benzenesulfonic or orthoor para-toluene-sulfonic acid.

Residues capable of elimination by hydrogenation are above all thecarbobenzoxy groups, such as the carbobenzoxy group andp-nitro-carbobenzoxy group, or paraphenylorpara-naphthyl-azobenzyloxy-carbonyl radicals which may be unsubstitutedor substituted by lower alkyl, halogen or nitro in the para-p0sition,such as the orangecolored para (para'-methoxy-phenylazo)benzyloxycarbonyl radical -(=MZ), or the p-phenylazo-benzyloxy-carbonylresidue, and also aralkyl residues such as the benzyl residue, thep-nitro-benzyl residue or p-halogen benzyl residues.

The temporary protection given to the guanidino group of the arginylresidue according to the present invention opens up new possibilities inthe synthesis of peptides, which do not involve the disadvantagesmentioned above.

The protected amino groups can be liberated selectively. By theacylation with the arylsulfonyl group subsequent acylation is prevented.Consequently only 'N -monoarylsulfonyl derivatives are obtained.

The arylsulfonyl radical can be eliminated with hydrolyzing agents, forexample by treatment with concentrated hydrochloric acid, or byhydrogenolysis, for example with sodium in liquid ammonia, or by othermethods known for eliminating the arylsulfonyl group.

In the course of the synthesis the substituent of the u-amino group canbe eliminated in the usual manner by hydrogenation. If the group whichprotects the u-amino group and which can be split off by hydrogenationis an acyl groupsuch as the carbobenzoxyor para-( am'- methoxyphenylazo)-benzyloxy-carbonyl group-it can alternatively be eliminated with a mildhydrolyzing agent without the arylsulfonyl radical in the guanidinogroup being eliminated too.

The introduction of the protective group and its elimination areperformed by as such known methods. An advantageous way to acylate the\guanidino group is by reaction with an arylsulionyl halide, for examplean arylsulfonyl chloride or bromide, such as benzeneor orthoorpara-toluene-sulfonyl chloride or bromide. The reaction isadvantageously conducted in a water-miscible organic solvent in thepresence of an alkali, for example in acetone in the presence of sodiumhydroxide solution. The reaction gives good yields.

The following examples illustrate the invention.

Example 1 3.43 grams of N"-carbobenzoxy-L-arginine are dissolved in 16.5cc. (6 equivalents) of cold 4 N-sodium hydroxide solution, and 100 cc.of acetone are added to this solution. The solution is treated in an icebath with 5.31 grams of para-toluenesulfonyl chloride (=tosyl chloride;about 2.5 equivalents) in small portions. After 1 hour the solution isdiluted with water, acidified with hydrochloric acid and extracted withethyl acetate. The ethyl acetate extract is extracted with diluteammonia, the resulting alkaline solution of N-carbobenzoxy-N -tosyl-L-arginine is acidified, again extracted with ethyl acetate, and theextract is evaporated. The residue, which still contains some unreactedN -carbobenzoxy-arginine, is dissolved in ethanol of 50% strength andpercolated through a small ion-exchanger column (Dowex 50, H+ form). Onevaporation, the eluates yield pure N -carbobenzoxy- N -tosyl-L-argininein the form of a glassy substance. Yield: 4.1 grams (81% of theory).Optical rotation [a] =+9.5 (in ethanol).

Example 2 To eliminate the carbobenzoxy group of N-carbobenzoxy-N-tosyl-L-arginine, this compound is subjected to catalytichydrogenation.

The arginine derivative is treated with hydrogen under atmosphericpressure in a methanolic solution containing 1 equivalent ofN-hydrochloric acid and a palladium catalyst.

The hydrochloride is isolated in the usual manner; it is a glassysubstance. Yield:

R value=0.36 in paper-chromatography in the system butanol+g1acialacetic acid-l-water (4:1:1). Spot test: ninhydrin positive, Sakaguchinegative.

Paper-electrophoresis at 200 volts and pH about 6.5 [solvent system:2:4:6-collidin-l-glacial acetic acid +water (8.9:3.l:8.8)] reveals asingle spot which is ninhydrin-positive and Sakaguchi-negative andmigrates towards the cathode at the same speed as leucine.

Example 3 Alternatively, the selective elimination of the carbobenzoxygroup of N-carbobenzoxy-N -tosyl-Irarginine can be performed bytreatment With a slight excess of 2 N-hydrogen bromide in glacial aceticacid for 1 hour at room temperature.

The hydrobromide is isolated in the known manner and forms a glassysubstance. Yield: 85%.

The paper-chromatography and electrophoresis are performed as describedin Example 2 and produce identical results.

Example 4 6.4 mg. of the hydrochloride of N -tosyl-L-arginine arestirred for 3 hours with cc. of water, 5 cc. of acetone and 1 gram ofmagnesium oxide. A solution of 600 mg. ofpara-(para'-methoxyphenylazo)-benzyloxycarbonyl chloride (=MZ- chloride)in 5 cc. of acetone is then added, and the mixture is stirred on for 18hours at room temperature.

N -Ml-N -tosyl-L-arginine is isolated as described for the carbobenzoxyderivative in Example 1. Yield: 885 mg. (=97% of theory). crystallinesubstance melting at 260 C. with decomposition. R value on acetytlatedpaper in the system butanol +glacial acetic acid-l-water (4:1:1)=0.61.On Whatman paper No. 1 [bottom phase: carbon tetrachloride+methanol+pyridine+Water (4:2:1)], R value=0.38.

The identical product is obtained by tosylating N- [para (paramethoxyphenylazo) benzyloxy-carbonyl]-L-arginine as described in Example1.

Example 5 750 mg. ofN"-[para-(para'-methoxyphenylazo)-benzyloXy-carbonyl]-N -tosyl-argininein ethyl acetate are mixed with a solution of 440 mg. ofL-tryptophyl-glycine methyl ester in ethyl acetate, whereupon theinsoluble salt separates out as an oil. The solvent is evaporated invacuo and the residue dissolved in 80 cc. of acetonitrile. The solutionis cooled to 0 C. and treated with 290 mg. of dicyclohexyl carbodiimide.After 24 hours, the dicyclohexyl-urea is filtered oh and the filtrateevaporated in vacuo. The residue is dissolved in ethyl acetate andtreated with hydrochloric acid and dilute ammonia to yield pureN-[para-(para-methoxyphenylazo)-benzyloxy-carbonyl] N tosyl arginyltryptophyl-glycine methyl ester.

Paper-chromatographic examination on acetylated paper reveals no spotsof positive reaction to ninhydrin or according to Sakaguchi but merelyan isolated orangecolored spot of the following R values:

With butanoH-glacial acetic acid+water (4:1:1)=0.701; Carbontetrachloride methanol pyridine Water (4:2:1rl), with the bottomphase:0.256; with the supernatant phase=0.630.

Example 6 The para- (para'-methoxyphenylazo) -benzyloxy-carbon- It is anorange-colored, microyl(=) group of N -MZ-N-tosyl-arginyl-tryptophyl-glycine methyl ester (Example 5) is eliminatedby catalytic hydrogenation in methanol-f-glacial acetic acid with apalladium catalyst.

The MZ-group of N -MZ-N -tosyl-arginyl-tryptophylglycine methyl estercan alternatively be eliminated by treatment With 3 N-hydrogen bromidein glacial acetic acid. The elimination is performed with a slightexcess of 3 N-hydrogen bromide in glacial acetic acid for 3 hours.

The resulting tripeptides obtained in either case possess identicalproperties.

R values on Whatman paper No. 1:

With butanol+glacial acetic acid-I-Water (4:1:l)=0.77

(ninhydn'n and Ehrlich tests positive);

With butanol pyridine glacial acetic acid water The identical tripeptideester (H.N -tosyl-arginyltryptophyl-glycine hydroxymethyl) is obtainedwhen N- [para-(para-methoxyphenylazo)-benzyloxy-carbonyl]-Ntosyl-arginine is replaced by N-carbobenzoxy-N -tosyliarginine.

What is claimed is:

1. u-Arnino-protected peptides containing an arginyl radical in Whichthe guanidino group is substituted by a p-toluenesulfonyl radical andthe u-amino group is substituted by a member selected from the groupconsisting of a carbobenzoxy group, a p-phenyl-azobenzyloxycarbonylgroup, a p-naphthyl-azobenzyloXy-carbonyl group, ap(p'-methoXy-phenylazo)carbonyl group, a benzyl group, a p-nitrobenzylgroup and a p-halogen benzyl group.

2. -N-carbobenzoxy-N -tosyl-L-arginine.

3. N -[p-(p-methoxy-phenylazo)-benzyloxy-carbonyl]- N -tosyl-L-arginine.

4. N tosylL-arginine.

5. N -tosyl-L-arginine-peptides.

References Cited in the file of this patent Barrass et al.: JournalChem. Society (London), Part III, pages 3134-39, July 1957.

Anson: Advances in Protein Chemistry, volume 5, pages 25-32, 1949(Academic Press Inc., publishers).

Clarke: Benzenesulfonylguanidines, J.A.C.S. 54, 1964- 68 (1932).

Schwyzer et al.: Helvetica Chimica Acta, volume 41, fasc. II, pages491-498, March 15, 1958.

Greenstein et al.: Chemistry of the Amino Acids, volume II, John Wileyand Sons, New York, 1961 (pages 883-901; 1068-1077 of interest).

SchWyzer et al.: Helvetica Chimica Acta, volume 40, fasc. IH, pages624-639, May 2, 1957.

Milne et al.: Journal of the American Chemical Society (1957), vol. 79,pages 639-644.

1. A-AMINO-PROTECTED PEPTIDES CONTAINING AN ARGINYL RADICAL IN WHICH THEGUANIDINO GROUP IS SUBSTITUTED BY A P-TOLUENESULFONYL RADICAL AND THEA-AMINO GROUP IS SUBSTITUTED BY A MEMBER SELECTED FROM THE GROUPCONSISTING OF A CARBOBENZOXY GROUP, A P-PHENYL-AZOBENZYLOXYCARBONYLGROUP, A P-NAPHTHYL-AZOBENZYLOXY-CARBONYL GROUP, AP-(P''-METHOXY-PHENYLAZO)CARBONYL GROUP, A BENZYL GROUP, A P-NITROBENZYLGROUP AND A P-HALOGEN BENZYL GROUP.
 4. NG-TOSYL-L-ARGININE. 5.NG-TOSYL-L-ARGININE-PEPTIDES.